Farnesoid x receptor modulators

ABSTRACT

The present invention provides a compound of formula (I):or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof, wherein R1, R2, R3, R4, R5, and R6 are as described herein. The present invention relates generally to selective FXR agonists and to methods of making and using them.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.17/228,819, filed Apr. 13, 2021, which is a continuation of U.S. patentapplication Ser. No. 16/695,528, filed Nov. 26, 2019, now U.S. Pat. No.11,000,532 which is a continuation of U.S. patent application Ser. No.16/124,139, filed Sep. 6, 2018, now U.S. Pat. No. 10,532,061, which is acontinuation of U.S. patent application Ser. No. 15/434,685, filed Feb.16, 2017, which is a continuation of U.S. patent application Ser. No.14/120,366, filed May 14, 2014, now U.S. Pat. No. 9,611,289, whichclaims priority to, and the benefit of, U.S. Provisional Application No.61/823,169, filed May 14, 2013, the entire contents of each of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

FXR is a member of the nuclear receptor family of ligand-activatedtranscription factors that includes receptors for the steroid, retinoid,and thyroid hormones (D. J. Mangelsdorf, et al., Cell 83:841-850(1995)). Northern and in situ analysis show that FXR is most abundantlyexpressed in the liver, intestine, kidney, and adrenal (B. M. Forman, etal., Cell 81:687-693 (1995) and W. Seol, et al., Mol. Endocrinnol.9:72-85 (1995)). FXR binds to DNA as a heterodimer with the 9-cisretinoic acid receptor (RXR). The rat FXR is activated by micromolarconcentrations of farnesoids such as farnesol and juvenile hormone (B.M. Forman, et al., Cell 81:687-693 (1995)). However, these compoundsfailed to activate the mouse and human FXR, leaving the nature of theendogenous FXR ligands in doubt. Several naturally-occurring bile acids(e.g., chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholicacid (LCA), and the taurine and glycine conjugates thereof) serve as FXRligands and bind to and activate FXR at physiological concentrations (WO00/37077).

Bile acids are cholesterol metabolites that are formed in the liver andsecreted into the duodenum of the intestine, where they have importantroles in the solubilization and absorption of dietary lipids andvitamins. Most bile acids (˜95%) are subsequently reabsorbed in theileum and returned to the liver via the enterohepatic circulatorysystem. The conversion of cholesterol to bile acids in the liver isunder feedback regulation: bile acids down-regulate the transcription ofcytochrome P450 7a (CYP7a), which encodes the enzyme that catalyzes therate limiting step in bile acid biosynthesis. It is suggested that FXRis involved in the repression of CYP7a expression by bile acids (D. W.Russell, Cell 97:539-542 (1999)). In the ileum, bile acids induce theexpression of the intestinal bile acid binding protein (IBABP), whichbinds bile acids with high affinity and may be involved in theircellular uptake and trafficking. It is demonstrated that bile acidsmediate their effects on IBABP expression through activation of FXR,which binds to an IR-1 type response element that is conserved in thehuman, rat, and mouse IBABP gene promoters. Thus, FXR is involved inboth the stimulation (IBABP) and the repression (CYP7a) of target genesinvolved in bile acid and cholesterol homeostasis. Accordingly, there isa need for FXR modulators suitable for drug development. The presentinvention addresses this need.

SUMMARY OF THE INVENTION

The invention provides compounds and methods of preparing thesecompounds. Specifically, the invention provides a compound of formula I:

or a pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof, wherein R¹, R², R³, R⁴, R⁵ and R⁶ are as described herein. Thecompounds of the invention are useful for treating and preventingdiseases and conditions.

The invention also provides a pharmaceutical composition comprising acompound of the invention or a pharmaceutically acceptable salt,solvate, or amino acid conjugate thereof, and a pharmaceuticallyacceptable carrier or excipient.

The invention also provides a method for the treatment or prevention ofa disease and condition, comprising administering to the subject in needthereof an effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate, or amino acid conjugatethereof. In one aspect, the disease or condition is FXR-mediated.

The invention also provides for the manufacture of a medicament fortreating or preventing a disease or condition (e.g., a disease orcondition mediated by FXR), wherein the medicament comprises a compoundof the invention or a pharmaceutically acceptable salt, solvate, oramino acid conjugate thereof.

The invention also provides a composition for use in a method fortreating or preventing a disease or condition (e.g., a disease orcondition mediated by FXR), wherein the composition comprises a compoundof the invention or a pharmaceutically acceptable salt, solvate, oramino acid conjugate thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the activity of a compound of the inventionand a comparison compound in a transactivation assay in HEK293T cells

FIG. 2 is a series of graphs showing the lack of TGR5 activity of acompound of the invention in human enteroendocrine cells expressing TGR5at physiological level (A) and in human Chinese hamster ovary (CHO)cells over-expressing TGR5 (B).

FIG. 3 is a series of graphs showing the activity of a compound of theinvention and other comparison compounds in regulating expression ofOSTα (A), OSTβ (B), BSEP (C), MRP2 (D), CYP7A1 (E), SHP (F), FGF-19 (G),and UGT2B4 (H).

FIG. 4 is a series of graphs showing the activity of a compound of theinvention and other comparison compounds in regulating PLTP involved inlipid metabolism (A), SREBP-1C (B), APOCII (C), and PPARγ (D).

FIG. 5 is a graph showing the regulation of a compound of the inventionand other comparison compounds on PEPCK gene.

FIG. 6 is a graph showing the measurement of ATP in HepG2 cells, treatedwith the indicated concentrations of a compound of the invention for 4h.

FIG. 7 is a series of graphs showing the choleretic effect of Compound100 for id and iv administration (A), the secretion of Compound 100 overtime for id and iv administration (B), and the plasma concentration ofCompound 100 over time (C).

DETAILED DESCRIPTION OF THE INVENTION Compounds of the Invention

The present invention relates to a compound of formula I:

or a pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof, wherein:

R¹ is hydroxyl;

R² is hydrogen, hydroxyl, alkyl, or halogen, wherein said alkyl isunsubstituted or substituted with one or more R^(a);

R³ is hydrogen, hydroxyl, alkyl, or halogen, wherein said alkyl isunsubstituted or substituted with one or more R^(b);

R⁴ is hydrogen, alkyl, alkenyl, alkynyl, or halogen, wherein said alkylis unsubstituted or substituted with one or more R^(c);

R^(a), R^(b), and R^(c) are each independently halogen or hydroxyl;

R⁵ is hydroxyl, OSO₃H, OSO₃ ⁻, OCOCH₃, OPO₃H, OPO₃ ²⁻, or hydrogen; and

R⁶ is hydroxyl, OSO₃H, OSO₃ ⁻, OCOCH₃, OPO₃H, OPO₃ ²⁻, or hydrogen;

or taken together R⁵ and R⁶ with the carbon atom to which they areattached form a carbonyl.

In one aspect, the present invention relates to a compound formula II:

or a pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof.

In one aspect, the present invention relates to a compound of formulaIII:

or a pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof.

In one aspect, the present invention relates to a compound of formulaIV:

or a pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein the compound is the compound (e.g., the nativecompound, or the compound in the non-salt, unsolvated, andnon-conjugated form).

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein the compound is the pharmaceutically acceptablesalt.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein the compound is the amino acid conjugate. In oneaspect, the amino acid conjugate is a glycine conjugate. In one aspect,the amino acid conjugate is a taurine conjugate.

In one aspect, the present invention relates to a compound of formula I,wherein one of R² or R³ is hydroxyl or halogen and the remaining R² orR³ is hydrogen or unsubstituted alkyl. In one aspect, one of R² or R³ ishydroxyl and the remaining R² or R³ is hydrogen.

In one aspect, the present invention relates to a compound of formula I,wherein one of R⁵ or R⁶ is hydroxyl and the remaining R⁵ or R⁶ ishydrogen.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein R² is hydroxyl or halogen. In one aspect, R² ishydroxyl. In another aspect, R² is halogen.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein R³ is hydrogen or unsubstituted alkyl. In oneaspect, R³ is hydrogen. In another aspect, R³ is methyl.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein R² is hydroxyl and R³ is hydrogen.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein R⁵ is hydroxyl.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein R⁶ is hydrogen.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein R² and R⁵ are each hydroxyl and R³ and R⁶ areeach hydrogen.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein R⁴ is alkyl or hydrogen. In one aspect, thepresent invention relates to a compound of formula I, II, III, or IV,wherein R⁴ is unsubstituted alkyl. In one aspect, R⁴ is methyl, ethyl,propyl, or butyl. In one aspect, R⁴ is methyl or ethyl. In one aspect,R⁴ is methyl. In one aspect, R⁴ is ethyl.

In one aspect, the present invention relates to compound

or a pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein the compound is an FXR agonist. In one aspect,the compound of the invention is a highly potent FXR agonist. Forexample, the compound of the invention activates FXR at a concentrationbelow 1 μM, below 0.8 μM, below 0.6 μM, below 0.4 μM, or below 0.2 μM(e.g., as measured by an AlphaScreen assay), as compared to 15 μM forCDCA. For example, the compound of the invention activates FXR at aconcentration below 0.2 μM (e.g., as measured by an AlphaScreen assay).For example, the compound of the invention activates FXR with an EC₅₀below 1 μM, below 0.8 μM, below 0.6 μM, below 0.4 μM, or below 0.2 μM(e.g., as measured by an AlphaScreen assay), as compared to 8.9 μM forCDCA. For example, the compound of the invention activates FXR with anEC₅₀ below 0.2 μM (e.g., as measured by an AlphaScreen assay).

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein the compound is not active against other nuclearreceptors. In one aspect, the present invention relates to a compound offormula I, II, III, or IV, wherein the compound does not activate TGR5(e.g., as measured by an HTR-FRET TGR5 assay, where the TGR5 is eitherexpressed at a physiological level or overexpressed).

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein the compound induces apoptosis.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein the compound shows no cytotoxic effect on humanHepG2 liver cells (e.g., as measured by an LDH release assay or anintracellular ATP assay).

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein the compound does not inhibit one or more CYP450isoforms selected from CYP1A2, CYP3A4 (green substrate), CYP3A4 (bluesubstrate), CYP2C9, CYP2C19, CYP2D6, and CYP2E1. For example, thecompounds of the invention have an IC₅₀ greater than 10 μM as measuredby CPY450 inhibition assay.

In one aspect, the present invention relates to a compound of formula I,II, III, or IV, wherein the compound does not inhibit the human ERGpotassium channel.

In one aspect, the present invention relates to a method of synthesizinga compound of the invention, or a pharmaceutically acceptable salt,solvate, or amino acid conjugate thereof.

In one aspect, the present invention relates to a kit containing one ormore compounds of the invention, or a pharmaceutically acceptable salt,solvate, or amino acid conjugate thereof. In one aspect, the kit furthercontains a pharmaceutically acceptable ingredient.

In one aspect, the present invention relates to a pharmaceuticalcomposition comprising a compound of the invention and apharmaceutically acceptable excipient.

One technical problem to be solved by the present invention is theidentification of novel compounds that are agonists of the nuclearhormone farnesoid X receptor (FXR), which represents an attractivetarget for the treatment of metabolic and chronic liver diseases. It iswell known that natural bile acids modulate not only several nuclearhormone receptors, but are also agonists for the G protein-coupledreceptor (GPCR) TGR5. Selectivity can be a problem for drug compoundsdirected to modulating a nuclear hormone receptor. It is therefore anobjective of the present invention to provide a compound that is aspecific FXR agonist, for example, a compound that shows no activityagainst other nuclear receptors or a compound that does not activate thebile acid GPCR TGR5. Other problems in the development of a drugcompound include a non-suitable pharmacokinetic profile, safety issuessuch as toxicity (e.g., liver) and undesirable drug-drug interactions.Accordingly, further objectives of the present invention are to providecompounds that do not suffer from the aforementioned technical problems,i.e., a compound that has a suitable pharmacokinetic profile, a compoundthat does not exert a cytotoxic effect on cells, a compound that doesnot inhibit cytochrome P450 enzymes, and/or a compound that does notinhibit hERG.

The patent and scientific literature referred to herein establishesknowledge that is available to those with skill in the art. The issuedpatents, applications, and references that are cited herein are herebyincorporated by reference to the same extent as if each was specificallyand individually indicated to be incorporated by reference. In the caseof inconsistencies, the present disclosure will prevail.

For purposes of the present invention, the following definitions will beused (unless expressly stated otherwise).

The general chemical terms used throughout have their usual meanings.For example, the term alkyl refers to a branched or unbranched saturatedhydrocarbon group. The term “n-alkyl” refers to an unbranched alkylgroup. The term “C_(x)-C_(y) alkyl” refers to an alkyl group havingbetween x and y carbon atoms, inclusively, in the branched or unbranchedhydrocarbon group. By way of illustration, but without limitation, theterm “C₁-C₈ alkyl” refers to a straight chain or branched hydrocarbonmoiety having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “C₁-C₆” refers toa straight chain or branched hydrocarbon moiety having 1, 2, 3, 4, 5, or6 carbon atoms. “C₁-C₄ alkyl” refers to a straight chain or branchedhydrocarbon moiety having 1, 2, 3, or 4 carbon atoms, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, andtert-butyl. The term “C₁-C₄ n-alkyl” refers to straight chainhydrocarbon moieties that have from 1, 2, 3, or 4 carbon atoms includingmethyl, ethyl, n-propyl, and n-butyl. The term “C₃-C₆ cycloalkyl” refersto cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term “C₃-C₇cycloalkyl” also includes cycloheptyl. The term “C₃-C₈ cycloalkyl” alsoincludes cyclooctyl. Cycloalkylalkyl refers to cycloalkyl moietieslinked through an alkyl linker chain, as for example, but withoutlimitation, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl,cyclopropylbutyl, cyclobutylmethyl, cyclobutylethyl, cyclobutylpropyl,cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl,cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Each alkyl,cycloalkyl, and cycloalkylalkyl group may be optionally substituted asspecified herein.

The term “C₄-C₈ cycloalkenyl” refers cyclobutenyl, cyclopentyl,cyclohexenyl, cycloheptenyl, and cyclooctenyl rings having one or moresites of unsaturation, e.g., one or more double bonds.

The term “halogen” refers to fluoro, chloro, bromo, or iodo.

The term “hydroxyl” means OH.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds unless otherwise specified. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. The permissible substituents can be one or more andthe same or different for appropriate organic compounds. For purposes ofthis invention, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms. Thisinvention is not intended to be limited in any manner by the permissiblesubstituents of organic compounds.

The term “pharmaceutical” or “pharmaceutically acceptable” when usedherein as an adjective, means substantially non-toxic and substantiallynon-deleterious to the recipient.

By “pharmaceutical formulation” it is further meant that the carrier,solvent, excipient, and salt must be compatible with the activeingredient of the formulation (e.g., a compound of the invention). It isunderstood by those of ordinary skill in this art that the terms“pharmaceutical formulation” and “pharmaceutical composition” aregenerally interchangeable, and they are so used for the purposes of thisapplication.

Suitable pharmaceutically acceptable salts according to the inventionwill be readily determined by one skilled in the art and will include,for example, basic salts such as alkali or alkaline-earth metallic saltsmade from aluminium, calcium, lithium, magnesium, potassium, sodium, andzinc or organic salts made from N,N′-dibenzylethylenediamine,chlorprocaine, choline, diethanolamine, ethylenediamine, meglumine(N-methylglucamine), and procaine. Salts with pharmaceuticallyacceptable amines such as lysine, arginine, tromethamine, triethylamineand the like can also be used. Such salts of the compounds of theinvention may be prepared using conventional techniques, from thecompound of the invention by reacting, for example, the appropriate basewith the compound of the invention.

When used in medicine, the salts of a compound of the invention shouldbe pharmaceutically acceptable, but pharmaceutically unacceptable saltsmay conveniently be used to prepare the corresponding free base orpharmaceutically acceptable salts thereof.

As used herein, the term “amino acid conjugate” refers to a conjugate ofa compound of the invention with any suitable amino acid. In one aspect,such suitable amino acid conjugate of a compound of the invention willhave the added advantage of enhanced integrity in bile or intestinalfluids. The present invention encompasses the glycine and taurineconjugates of any of the compounds of the invention. For example, theglycine and the taurine conjugates of a compound of formula I have thefollowing formula:

In one aspect, the glycine and taurine conjugates of a compound of theinvention may be a pharmaceutically acceptable salt thereof. The aminoacid conjugates of compounds of the invention can be prepared accordingto methods known in the art. For example, the free acid can be coupledto the glycine or taurine amino acid using standard peptide couplingconditions.

In one aspect, the sodium salt of the taurine conjugate of Compound 100can be prepared as follows.

Compound 100 is treated with a base (e.g., Et₃N) and taurine in a polarprotic solvent (e.g., EtOH). The resulting mixture can be treated with acoupling reagent (e.g., DMT-MM(4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride)).The reaction mixture can be concentrated and dissolved in a base (e.g.,3% w/v aqueous solution of NaOH). The resulting reaction mixture can beextracted with an organic solvent (e.g., AcOEt). The aqueous phase canbe concentrated and filtered on a silica pad, eluting first with, e.g.,H₂O (until neutral pH) and then with, e.g., H₂O/MeOH 80:20 v/v to givethe taurine conjugate of Compound 100. Suitable amino acids include butare not limited to glycine and taurine.

Some of the compounds of the present invention may exist in unsolvatedas well as solvated forms such as, for example, hydrates.

The present invention provides methods for the synthesis of thecompounds of invention described herein. The present invention alsoprovides detailed methods for the synthesis of various disclosedcompounds of the invention according to the following schemes as shownin the examples.

The synthetic processes of the invention can tolerate a wide variety offunctional groups, therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or prodrug thereof.

Compounds of the invention can be prepared in a variety of ways usingcommercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5th edition, John Wiley & Sons: New York, 2001; andGreene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3rd edition, John Wiley & Sons: New York, 1999, incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentinvention.

All the abbreviations used in this application are found in “ProtectiveGroups in Organic Synthesis” by John Wiley & Sons, Inc, or the MERCKINDEX by MERCK & Co., Inc, or other chemistry books or chemicalscatalogs by chemicals vendor such as Aldrich, or according to usage knowin the art.

Reagents and conditions: a) 1) MeOH, p-TSA, ultrasound, 3 h,quantitative; 2) Ac₂O, NaHCO₃, THF, reflux 12 h, 85%; b) PCC, CH₂Cl₂, 6h, 62%; c) Ac₂O, Bi(OTf₃, CH₂Cl₂, 1 h, 91%; d) Br₂, Benzene, 30° C.overnight, 74%; e) NaBH₄, NaOAc, Pyr, r.t. 2 days, 80%; f) HI 57%, AcOH,r.t. 30 min; g) CrO₃, AcOH, r.t. 45 min; h) Zn dust, NaOAc, reflux 20min; i) NaOH 2M, MeOH, r.t. overnight, 65% from compound 5; l) NaBH₄,THF/H₂O 4:1, 70%; m) Na(s), sec-BuOH, 50° C., 70%.

The synthesis is based on the use of 6α-ethyl-cholic acid (6-ECA, 1) asstarting material which was prepared using methods known in the art.6-ECA (1) was treated with p-TSA in MeOH under ultrasound irradiation togive the corresponding methyl ester, which was selectively protected atthe C3 position by refluxing with Ac₂O in the present of NaHCO₃ in THFto afford compound 2. Treating compound 2 with PCC in CH₂Cl₂ at roomtemperature followed by treatment with Ac₂O, Bi(OTf)₃ in CH₂Cl₂ at roomtemperature afforded the intermediate methyl3α,7α-diacetoxy-12-oxo-5β-cholan-24-oate (compound 3; about 48% fromcompound 2).

Treatment of compound 3 with Br₂ in benzene for e.g., 12 h yieldedcompound 4. Reaction of compound 4 with NaBH₄ and NaOAc in freshlydistilled pyridine gave the corresponding 11β-12β epoxide (compound 5),in about 59% yield after silica gel purification. The reaction ofcompound 5 with HI in AcOH at room temperature afforded the halohydrineintermediate which were then oxidized at C11 position with CrO₃ in AcOHto generate compound 6. Reaction of compound 6 with Zn dust in boilingAcOH and alkali hydrolysis (NaOH/MeOH) afforded3α,7α-hydroxy-12-keto-5b-cholan-24-oic acid (compound 7; about 65% yieldfrom compound 5).

Compound 7 was stereoselectively reduced at the C11-carbonyl using NaBH₄in a mixture of THF/H₂O═ (4:1, v/v) to give3α,7α,11β-trihydroxy-6α-ethyl-5β-cholan-24-oic acid (Compound 100; about27% from compound 3), after chromatographic purification to affordCompound 100. Alternatively, compound 7 was reduced with sodium insec-BuOH at 50° C. to give Compound 101 (about 70% yield), afterpurification.

“Solvate” means a solvent addition form that contains either astoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

The term “suitable solvent” refers to any solvent, or mixture ofsolvents, inert to the ongoing reaction that sufficiently solubilizesthe reactants to afford a medium within which to effect the desiredreaction.

The compounds described herein can have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom canbe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and can be isolated as a mixture of isomers or as separateisomeric forms. All chiral, diastereomeric, racemic, and geometricisomeric forms of a structure are intended, unless specificstereochemistry or isomeric form is specifically indicated. Allprocesses used to prepare compounds of the present invention andintermediates made therein are considered to be part of the presentinvention. All tautomers of shown or described compounds are alsoconsidered to be part of the present invention. Furthermore, theinvention also includes metabolites of the compounds described herein.

The invention also comprehends isotopically-labeled compounds, which areidentical to those recited in the formulae of the invention, but for thefact that one or more atoms are replaced by an atom having an atomicmass or mass number different from the atomic mass or mass number mostcommonly found in nature. Examples of isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, fluorine, such as ³H, ¹¹C, ¹⁴C, ²H, and ¹⁸F.

Compounds of the present invention and pharmaceutically acceptablesalts, solvates or amino acid conjugates thereof that contain theaforementioned isotopes and/or other isotopes of other atoms are withinthe scope of the present invention. Isotopically-labeled compounds ofthe present invention, for example those into which radioactive isotopessuch as ³H and ¹⁴C are incorporated, are useful in drug and/or substratetissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e.,¹⁴C, isotopes are particularly preferred for their ease of preparationand detectability. ¹¹C and ¹⁸F isotopes are particularly useful in PET(positron emission tomography). PET is useful in brain imaging. Further,substitution with heavier isotopes such as deuterium, i.e., ²H, canafford certain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements and, hence, may be preferred in some circumstances.Isotopically labeled compounds of this invention can generally beprepared through techniques known in the art, such as by carrying outthe procedures disclosed in the Schemes and/or in the Examples below, bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. In one embodiment, the compounds ofthe invention, salts, hydrates, solvates, or amino acid conjugatesthereof are not isotopically labelled.

When any variable (e.g., R^(x)) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with one or more R^(X)moieties, then R^(X) at each occurrence is selected independently fromthe definition of R^(X). Also, combinations of substituents and/orvariables are permissible, but only if such combinations result instable compounds within a designated atom's normal valency.

As used herein, the term “treat,” “treating,” or “treatment” is meantdecreasing the symptoms, markers, and/or any negative effects of acondition in any appreciable degree in a subject who currently has thecondition. In some embodiments, treatment may be administered to asubject who exhibits only early signs of the condition for the purposeof decreasing the risk of developing the disease or condition.

As used herein, the term “prevent,” “prevention,” or “preventing” refersto any method to partially or completely prevent or delay the onset ofone or more symptoms or features of a disease, disorder, and/orcondition. Prevention may be administered to a subject who does notexhibit signs of a disease or condition.

As used herein, “subject” means a human or animal (in the case of ananimal, more typically a mammal). In one aspect, the subject is a human.Such subject can be considered to be in need of treatment with an FXRagonist.

As used herein, “unsaturated” refers to compounds or structures havingat least one degree of unsaturation (e.g., at least one double or triplebond).

As used herein, the term “a compound of the invention” includes acompound of any of formulae I, II, III, or IV, or any compoundexplicitly disclosed herein.

As used herein, farnesoid X receptor or FXR refers to all mammalianforms of such receptor including, for example, alternative spliceisoforms and naturally occurring isoforms (see, e.g., Huber et al., Gene290:35-43 (2002)). Representative FXR species include, withoutlimitation rat FXR (Gen Bank Accession No. NM_021745), mouse FXR(Genbank Accession No. NM_009108), and human FXR (GenBank Accession No.NM_005123).

As used herein, Compound A is

which is also known as obeticholic acid, INT-747, 6ECDCA, 6-alpha-ethylchenodeoxycholic acid, or 6α-ethyl-3α,7α-dihydroxy-5β-cholan-24-oicacid.

As used herein, Compound B is

which is also known as INT-767 or6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulfate sodium salt.

As used herein, Compound C is

which is also known as INT-777 or 6α-ethyl-23(S)-methyl-3α,7α,12αtrihydroxy-5β-cholan-24-oic acid.

As used herein, Compound D is

which is also known as 6α-ethyl-23(R)-methyl chenodeoxycholic acid, andS-EMCDCA.

As used herein, Compound E is

As used herein, cholic acid is

which is also known as CA.

As used herein, chenodeoxycholic acid is

which is also known as CDCA.

As used herein, ursodeoxycholic acid is

which is also known as UDCA.

As used herein, taurochenodeoxycholic acid is

which is also known asn TCDCA.

As used herein, tauroursodeoxycholic acid is

which is also known as TUDCA.

As used herein, lithocholic acid is

which is also known as LCA.

Methods of the Invention

Compounds of the invention are useful in therapy in subjects such asmammals, including humans. In particular, compounds of the invention areuseful in a method of treating or preventing a disease or condition in asubject comprising administering to the subject in need thereof aneffective amount of a compound of the invention or a pharmaceuticallyacceptable salt, solvate, or amino acid conjugate thereof. In oneaspect, the disease or condition is FXR-mediated (e.g., FXR plays a rolein the initiation or progress of the disease or condition). In oneaspect, the disease or condition is mediated by decreased FXR activity.In one aspect, the disease or condition is selected from cardiovasculardisease, chronic liver disease, lipid disorder, gastrointestinaldisease, renal disease, metabolic disease, cancer, and neurologicaldisease.

In one aspect, the invention relates to a method of treating orpreventing cardiovascular disease in a subject, comprising administeringto the subject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating cardiovascular disease. In one aspect, the invention relates toa method of preventing cardiovascular disease. In one aspect,cardiovascular disease selected from atherosclerosis, arteriosclerosis,dyslipidemia, hypercholesteremia, hyperlipidemia, hyperlipoproteinemia,and hypertriglyceridemia.

The term “hyperlipidemia” refers to the presence of an abnormallyelevated level of lipids in the blood. Hyperlipidemia can appear in atleast three forms: (1) hypercholesterolemia, i.e., an elevatedcholesterol level; (2) hypertriglyceridemia, i.e., an elevatedtriglyceride level; and (3) combined hyperlipidemia, i.e., a combinationof hypercholesterolemia and hypertriglyceridemia.

The term “dyslipidemia” refers to abnormal levels of lipoproteins inblood plasma including both depressed and/or elevated levels oflipoproteins (e.g., elevated levels of LDL, VLDL and depressed levels ofHDL).

In one aspect, the invention relates to a method selected from reducingcholesterol levels or modulating cholesterol metabolism, catabolism,absorption of dietary cholesterol, and reverse cholesterol transport ina subject, comprising administering to the subject in need thereof aneffective amount of a compound of the invention or a pharmaceuticallyacceptable salt, solvate, or amino acid conjugate thereof.

In one aspect, the invention relates to a method of treating orpreventing a disease affecting cholesterol, triglyceride, or bile acidlevels in a subject, comprising administering to the subject in needthereof an effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate, or amino acid conjugatethereof.

In one aspect, the invention relates to a method of loweringtriglycerides in a subject, comprising administering to the subject inneed thereof an effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate, or amino acid conjugatethereof.

In one aspect, the invention relates to a method of treating orpreventing a disease state associated with an elevated cholesterol levelin a subject, comprising administering to the subject in need thereof aneffective amount of a compound of the invention or a pharmaceuticallyacceptable salt, solvate, or amino acid conjugate thereof. In oneaspect, the invention relates to a method of treating a disease stateassociated with an elevated cholesterol level in a subject. In oneaspect, the invention relates to a method of preventing a disease stateassociated with an elevated cholesterol level in a subject. In oneaspect, the disease state is selected from coronary artery disease,angina pectoris, carotid artery disease, strokes, cerebralarteriosclerosis, and xanthoma.

In one aspect, the invention relates to a method of treating orpreventing a lipid disorder in a subject, comprising administering tothe subject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating a lipid disorder. In one aspect, the invention relates to amethod of preventing a lipid disorder.

Lipid disorders are the term for abnormalities of cholesterol andtriglycerides. Lipid abnormalities are associated with an increased riskfor vascular disease, and especially heart attacks and strokes.Abnormalities in lipid disorders are a combination of geneticpredisposition as well as the nature of dietary intake. Many lipiddisorders are associated with being overweight. Lipid disorders may alsobe associated with other diseases including diabetes, the metabolicsyndrome (sometimes called the insulin resistance syndrome), underactivethyroid or the result of certain medications (such as those used foranti-rejection regimens in people who have had transplants).

In one aspect, the invention relates to a method of treating orpreventing one or more symptoms of disease affecting lipid metabolism(i.e., lipodystrophy) in a subject, comprising administering to thesubject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating one or more symptoms of a disease affecting lipid metabolism.In one aspect, the invention relates to a method of preventing one ormore symptoms of a disease affecting lipid metabolism.

In one aspect, the invention relates to a method of decreasing lipidaccumulation in a subject, comprising administering to the subject inneed thereof an effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate, or amino acid conjugatethereof.

In one aspect, the invention relates to a method of treating orpreventing chronic liver disease in a subject, comprising administeringto the subject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating chronic liver disease. In one aspect, the invention relates toa method of preventing chronic liver disease. In one aspect, the chronicliver disease is selected from primary biliary cirrhosis (PBC),cerebrotendinous xanthomatosis (CTX), primary sclerosing cholangitis(PSC), drug induced cholestasis, intrahepatic cholestasis of pregnancy,parenteral nutrition associated cholestasis (PNAC), bacterial overgrowthor sepsis associated cholestasis, autoimmune hepatitis, chronic viralhepatitis, alcoholic liver disease, nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), liver transplantassociated graft versus host disease, living donor transplant liverregeneration, congenital hepatic fibrosis, choledocholithiasis,granulomatous liver disease, intra- or extrahepatic malignancy,Sjogren's syndrome, Sarcoidosis, Wilson's disease, Gaucher's disease,hemochromatosis, and alpha 1-antitrypsin deficiency.

In one aspect, the invention relates to a method of treating orpreventing one or more symptoms of cholestasis, including complicationsof cholestasis in a subject, comprising administering to the subject inneed thereof an effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate, or amino acid conjugatethereof. In one aspect, the invention relates to a method of treatingone or more symptoms of cholestasis. In one aspect, the inventionrelates to preventing one or more symptoms of cholestasis.

Cholestasis is typically caused by factors within the liver(intrahepatic) or outside the liver (extrahepatic) and leads to theaccumulation of bile salts, bile pigment bilirubin, and lipids in theblood stream instead of being eliminated normally. Intrahepaticcholestasis is characterized by widespread blockage of small ducts or bydisorders, such as hepatitis, that impair the body's ability toeliminate bile. Intrahepatic cholestasis may also be caused by alcoholicliver disease, primary biliary cirrhosis, cancer that has spread(metastasized) from another part of the body, primary sclerosingcholangitis, gallstones, biliary colic and acute cholecystitis. It canalso occur as a complication of surgery, serious injury, cysticfibrosis, infection, or intravenous feeding or be drug induced.Cholestasis may also occur as a complication of pregnancy and oftendevelops during the second and third trimesters.

Extrahepatic cholestasis is most often caused by choledocholithiasis(Bile Duct Stones), benign biliary strictures (non-cancerous narrowingof the common duct), cholangiocarcinoma (ductal carcinoma) andpancreatic carcinoma. Extrahepatic cholestasis can occur as a sideeffect of many medications.

A compound of the invention may be used for treating or preventing oneor more symptoms of intrahepatic or extrahepatic cholestasis, includingwithout limitation, biliary artesia, obstetric cholestasis, neonatalcholestasis, drug induced cholestasis, cholestasis arising fromHepatitis C infection, chronic cholestatic liver disease such as primarybiliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC).

In one aspect, the invention relates to a method of enhancing liverregeneration in a subject, comprising administering to the subject inneed thereof an effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate, or amino acid conjugatethereof. In one aspect, the method is enhancing liver regeneration forliver transplantation.

In one aspect, the invention relates to a method of treating orpreventing fibrosis in a subject, comprising administering to thesubject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating fibrosis. In one aspect, the invention relates to a method ofpreventing fibrosis.

Accordingly, as used herein, the term fibrosis refers to all recognizedfibrotic disorders, including fibrosis due to pathological conditions ordiseases, fibrosis due to physical trauma (“traumatic fibrosis”),fibrosis due to radiation damage, and fibrosis due to exposure tochemotherapeutics. As used herein, the term “organ fibrosis” includesbut is not limited to liver fibrosis, fibrosis of the kidneys, fibrosisof lung, and fibrosis of the intestine. “Traumatic fibrosis” includesbut is not limited to fibrosis secondary to surgery (surgical scarring),accidental physical trauma, burns, and hypertrophic scarring.

As used herein, “liver fibrosis” includes liver fibrosis due to anycause, including but not limited to virally-induced liver fibrosis suchas that due to hepatitis B or C virus; exposure to alcohol (alcoholicliver disease), certain pharmaceutical compounds including but notlimited to methotrexate, some chemotherapeutic agents, and chronicingestion of arsenicals or vitamin A in megadoses, oxidative stress,cancer radiation therapy or certain industrial chemicals including butnot limited to carbon tetrachloride and dimethylnitrosamine; anddiseases such as primary biliary cirrhosis, primary sclerosingcolangitis, fatty liver, obesity, non-alcoholic steatohepatitis, cysticfibrosis, hemochromatosis, auto-immune hepatitis, and steatohepatitis.Current therapy in liver fibrosis is primarily directed at removing thecausal agent, e.g., removing excess iron (e.g., in the case ofhemochromatosis), decreasing viral load (e.g., in the case of chronicviral hepatitis), or eliminating or decreasing exposure to toxins (e.g.,in the case of alcoholic liver disease). Anti-inflammatory drugs such ascorticosteroids and colchicine are also known for use in treatinginflammation that can lead to liver fibrosis.

As is known in the art, liver fibrosis may be clinically classified intofive stages of severity (S0, S1, S2, S3, and S4), usually based onhistological examination of a biopsy specimen. S0 indicates no fibrosis,whereas S4 indicates cirrhosis. While various criteria for staging theseverity of liver fibrosis exist, in general early stages of fibrosisare identified by discrete, localized areas of scarring in one portal(zone) of the liver, whereas later stages of fibrosis are identified bybridging fibrosis (scarring that crosses zones of the liver).

In one aspect, the invention relates to a method of treating orpreventing organ fibrosis in a subject, comprising administering to thesubject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the fibrosis is liver fibrosis.

In one aspect, the invention relates to a method of treating orpreventing gastrointestinal disease in a subject, comprisingadministering to the subject in need thereof an effective amount of acompound of the invention or a pharmaceutically acceptable salt,solvate, or amino acid conjugate thereof. In one aspect, the inventionrelates to a method of treating gastrointestinal disease. In one aspect,the invention relates to a method of preventing gastrointestinaldisease. In one aspect, the gastrointestinal disease is selected frominflammatory bowel disease (IBD), irritable bowel syndrome (IBS),bacterial overgrowth, malabsorption, post-radiation colitis, andmicroscopic colitis. In one aspect, the inflammatory bowel disease isselected from Crohn's disease and ulcerative colitis.

In one aspect, the invention relates to a method of treating orpreventing renal disease in a subject, comprising administering to thesubject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating renal disease. In one aspect, the invention relates to a methodof preventing renal disease. In one aspect, the renal disease isselected from diabetic nephropathy, focal segmental glomerulosclerosis(FSGS), hypertensive nephrosclerosis, chronic glomerulonephritis,chronic transplant glomerulopathy, chronic interstitial nephritis, andpolycystic kidney disease.

In one aspect, the invention relates to a method of treating orpreventing metabolic disease in a subject, comprising administering tothe subject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating renal disease. In one aspect, the invention relates to a methodof preventing renal disease. In one aspect, the metabolic disease isselected from insulin resistance, hyperglycemia, diabetes mellitus,diabesity, and obesity. In one aspect, the diabetes mellitus is type Idiabetes. In one aspect, the diabetes mellitus is type II diabetes.

Diabetes mellitus, commonly called diabetes, refers to a disease orcondition that is generally characterized by metabolic defects inproduction and utilization of glucose which result in the failure tomaintain appropriate blood sugar levels in the body.

In the case of type II diabetes, the disease is characterized by insulinresistance, in which insulin loses its ability to exert its biologicaleffects across a broad range of concentrations. This resistance toinsulin responsiveness results in insufficient insulin activation ofglucose uptake, oxidation and storage in muscle and inadequate insulinrepression of lipolysis in adipose tissue and of glucose production andsecretion in liver. The resulting condition is elevated blood glucose,which is called “hyperglycemia”. Uncontrolled hyperglycemia isassociated with increased and premature mortality due to an increasedrisk for microvascular and macrovascular diseases, including retinopathy(the impairment or loss of vision due to blood vessel damage in theeyes); neuropathy (nerve damage and foot problems due to blood vesseldamage to the nervous system); and nephropathy (kidney disease due toblood vessel damage in the kidneys), hypertension, cerebrovasculardisease and coronary heart disease. Therefore, control of glucosehomeostasis is a critically important approach for the treatment ofdiabetes.

Insulin resistance has been hypothesized to unify the clustering ofhypertension, glucose intolerance, hyperinsulinemia, increased levels oftriglyceride and decreased HDL cholesterol, and central and overallobesity. The association of insulin resistance with glucose intolerance,an increase in plasma triglyceride and a decrease in high-densitylipoprotein cholesterol concentrations, hypertension, hyperuricemia,smaller denser low-density lipoprotein particles, and higher circulatinglevels of plasminogen activator inhibitor-1, has been referred to as“Syndrome X”. Accordingly, methods of treating or preventing anydisorders related to insulin resistance including the cluster of diseasestates, conditions or disorders that make up “Syndrome X” are provided.In one aspect, the invention relates to a method of treating orpreventing metabolic syndrome in a subject, comprising administering tothe subject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating metabolic syndrome. In one aspect, the invention relates to amethod of preventing metabolic syndrome.

In one aspect, the invention relates to a method of treating orpreventing cancer in a subject, comprising administering to the subjectin need thereof an effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate, or amino acid conjugatethereof. In one aspect, the invention relates to a method of treatingcancer. In one aspect, the invention relates to a method of preventingcancer. In one aspect, the cancer is colorectal cancer.

In one aspect, the invention relates to a method of treating orpreventing gallstones in a subject, comprising administering to thesubject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating gallstones. In one aspect, the invention relates to a method ofpreventing gallstones.

A gallstone is a crystalline concretion formed within the gallbladder byaccretion of bile components. These calculi are formed in thegallbladder but may distally pass into other parts of the biliary tractsuch as the cystic duct, common bile duct, pancreatic duct, or theampulla of Vater. Rarely, in cases of severe inflammation, gallstonesmay erode through the gallbladder into adherent bowel potentiallycausing an obstruction termed gallstone ileus. Presence of gallstones inthe gallbladder may lead to acute cholecystitis, an inflammatorycondition characterized by retention of bile in the gallbladder andoften secondary infection by intestinal microorganisms, predominantlyEscherichia coli and Bacteroides species. Presence of gallstones inother parts of the biliary tract can cause obstruction of the bileducts, which can lead to serious conditions such as ascendingcholangitis or pancreatitis.

In one aspect, the invention relates to a method of treating orpreventing cholesterol gallstone disease in a subject, comprisingadministering to the subject in need thereof an effective amount of acompound of the invention or a pharmaceutically acceptable salt,solvate, or amino acid conjugate thereof. In one aspect, the inventionrelates to a method of treating cholesterol gallstone disease. In oneaspect, the invention relates to a method of preventing cholesterolgallstone disease.

In one aspect, the invention relates to a method of treating orpreventing neurological disease in a subject, comprising administeringto the subject in need thereof an effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, or amino acidconjugate thereof. In one aspect, the invention relates to a method oftreating neurological disease. In one aspect, the invention relates to amethod of preventing neurological disease. In one aspect, theneurological disease is stroke.

In one aspect, the invention relates to a method as described herein andfurther wherein, the compound is administered by a route selected fromoral, parenteral, intramuscular, intranasal, sublingual, intratracheal,inhalation, ocular, vaginal, rectal, and intracerebroventricular. In oneaspect, the route is oral.

In one aspect, the compound utilized in one or more of the methodsdescribed herein is an FXR agonist. In one aspect, the compound is aselective FXR agonist. In one aspect, the compound does not activateTGR5. In one aspect, the compound does not activate other nuclearreceptors involved in metabolic pathways (e.g., as measured by anAlphaScreen assay). In one aspect, such other nuclear receptors involvedin metabolic pathways are selected from LXRβ, PXR, CAR, PPARα, PPARδ,RARα, VDR, TR, PR, RXR, GR, and ER. In one aspect, the compound inducesapoptosis.

In one aspect, the invention relates to a method of regulating theexpression level of one or more genes involved in bile acid homeostasis.

In one aspect, the invention relates to a method of down regulating theexpression level of one or more genes selected from CYP7α1 and SREBP-1Cin a cell by administering to the cell a compound of the invention. Inone aspect, the invention relates to a method of up regulating theexpression level of one or more genes selected from OSTα, OSTβ, BSEP,SHP, UGT2B4, MRP2, FGF-19, PPARγ, PLTP, APOCII, and PEPCK in a cell byadministering to the cell a compound of the invention.

The invention also relates to the manufacture of a medicament fortreating or preventing a disease or condition (e.g., a disease orcondition mediated by FXR), wherein the medicament comprises a compoundof the invention or a pharmaceutically acceptable salt, solvate, oramino acid conjugate thereof. In one aspect, the invention relates tothe manufacture of a medicament for treating or preventing any one ofthe diseases or conditions described herein above, wherein themedicament comprises a compound of the invention or a pharmaceuticallyacceptable salt, solvate, or amino acid conjugate thereof.

The invention also relates to a composition for use in a method fortreating or preventing a disease or condition (e.g., a disease orcondition mediated by FXR), wherein the composition comprises a compoundof the invention or a pharmaceutically acceptable salt, solvate, oramino acid conjugate thereof. In one aspect, the invention relates to acomposition for use in a method for treating or preventing any one ofthe diseases or conditions described herein above, wherein thecomposition comprises a compound of the invention or a pharmaceuticallyacceptable salt, solvate, or amino acid conjugate thereof.

Formulations

The methods of the invention comprise the step of administering aneffective amount of a compound of the invention. As used herein, theterm an “effective amount” refers to an amount of a compound of theinvention which is sufficient to achieve the stated effect. Accordingly,an effective amount of a compound of the invention used in a method forthe prevention or treatment of FXR mediated diseases or conditions willbe an amount sufficient to prevent or treat the FXR mediated disease orcondition.

Similarly, an effective amount of a compound of the invention for use ina method for the prevention or treatment of a cholestatic liver diseaseor increasing bile flow will be an amount sufficient to increase bileflow to the intestine.

The amount of the compound of the invention which is required to achievethe desired biological effect will depend on a number of factors such asthe use for which it is intended, the means of administration, and therecipient, and will be ultimately at the discretion of the attendantphysician or veterinarian. In general, a typical daily dose for thetreatment of a FXR mediated disease and condition, for instance, may beexpected to lie in the range of from about 0.01 mg/kg to about 100mg/kg. This dose may be administered as a single unit dose or as severalseparate unit doses or as a continuous infusion. Similar dosages wouldbe applicable for the treatment of other diseases, conditions andtherapies including the prevention and treatment of cholestatic liverdiseases.

Thus, in a further aspect, the present invention provides apharmaceutical composition comprising, as active ingredient, a compoundof the invention together, and/or in admixture, with at least onepharmaceutical carrier or diluent. These pharmaceutical compositions maybe used in the prevention or treatment of the foregoing diseases orconditions.

The carrier must be pharmaceutically acceptable and must be compatiblewith, i.e. not have a deleterious effect upon, the other ingredients inthe composition. The carrier may be a solid or liquid and is preferablyformulated as a unit dose formulation, for example, a tablet which maycontain from 0.05 to 95% by weight of the active ingredient. If desired,other physiologically active ingredients may also be incorporated in thepharmaceutical compositions of the invention.

Possible formulations include those suitable for oral, sublingual,buccal, parenteral (for example subcutaneous, intramuscular, orintravenous), rectal, topical including transdermal, intranasal andinhalation administration. Most suitable means of administration for aparticular patient will depend on the nature and severity of the diseaseor condition being treated or the nature of the therapy being used andon the nature of the active compound, but where possible, oraladministration is preferred for the prevention and treatment of FXRmediated diseases and conditions. Formulations suitable for oraladministration may be provided as discrete units, such as tablets,capsules, cachets, lozenges, each containing a predetermined amount ofthe active compound; as powders or granules; as solutions or suspensionsin aqueous or non-aqueous liquids; or as oil-in-water or water-in-oilemulsions.

Formulations suitable for sublingual or buccal administration includelozenges comprising the active compound and, typically a flavoured base,such as sugar and acacia or tragacanth and pastilles comprising theactive compound in an inert base, such as gelatine and glycerine orsucrose acacia.

Formulations suitable for parenteral administration typically comprisesterile aqueous solutions containing a predetermined concentration ofthe active compound; the solution is preferably isotonic with the bloodof the intended recipient.

Additional formulations suitable for parenteral administration includeformulations containing physiologically suitable co-solvents and/orcomplexing agents such as surfactants and cyclodextrins. Oil-in-wateremulsions are also suitable formulations for parenteral formulations.Although such solutions are preferably administered intravenously, theymay also be administered by subcutaneous or intramuscular injection.

Formulations suitable for rectal administration are preferably providedas unit-dose suppositories comprising the active ingredient in one ormore solid carriers forming the suppository base, for example, cocoabutter.

Formulations suitable for topical or intranasal application includeointments, creams, lotions, pastes, gels, sprays, aerosols and oils.Suitable carriers for such formulations include petroleum jelly,lanolin, polyethyleneglycols, alcohols, and combinations thereof.

Formulations of the invention may be prepared by any suitable method,typically by uniformly and intimately admixing the active compound withliquids or finely divided solid carriers or both, in the requiredproportions and then, if necessary, shaping the resulting mixture intothe desired shape.

For example a tablet may be prepared by compressing an intimate mixturecomprising a powder or granules of the active ingredient and one or moreoptional ingredients, such as a binder, lubricant, inert diluent, orsurface active dispersing agent, or by moulding an intimate mixture ofpowdered active ingredient and inert liquid diluent.

Suitable formulations for administration by inhalation include fineparticle dusts or mists which may be generated by means of various typesof metered dose pressurised aerosols, nebulisers, or insufflators.

For pulmonary administration via the mouth, the particle size of thepowder or droplets is typically in the range 0.5-10 μm, preferably 1-5μm, to ensure delivery into the bronchial tree. For nasaladministration, a particle size in the range 10-500 μm is preferred toensure retention in the nasal cavity.

Metered dose inhalers are pressurised aerosol dispensers, typicallycontaining a suspension or solution formulation of the active ingredientin a liquefied propellant. During use, these devices discharge theformulation through a valve adapted to deliver a metered volume,typically from 10 to 150 μl, to produce a fine particle spray containingthe active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one or more co-solvents, forexample, ethanol surfactants, such as oleic acid or sorbitan trioleate,anti-oxidants and suitable flavouring agents.

Nebulisers are commercially available devices that transform solutionsor suspensions of the active ingredient into a therapeutic aerosol misteither by means of acceleration of a compressed gas typically air oroxygen, through a narrow venturi orifice, or by means of ultrasonicagitation. Suitable formulations for use in nebulisers consist of theactive ingredient in a liquid carrier and comprise up to 40% w/w of theformulation, preferably less than 20% w/w. The carrier is typicallywater or a dilute aqueous alcoholic solution, preferably made isotonicwith body fluids by the addition of, for example, sodium chloride.Optional additives include preservatives if the formulation is notprepared sterile, for example, methyl hydroxy-benzoate, anti-oxidants,flavouring agents, volatile oils, buffering agents and surfactants.

Suitable formulations for administration by insufflation include finelycomminuted powders which may be delivered by means of an insufflator ortaken into the nasal cavity in the manner of a snuff. In theinsufflator, the powder is contained in capsules or cartridges,typically made of gelatin or plastic, which are either pierced or openedin situ and the powder delivered by air drawn through the device uponinhalation or by means of a manually-operated pump. The powder employedin the insufflator consists either solely of the active ingredient or ofa powder blend comprising the active ingredient, a suitable powderdiluent, such as lactose, and an optional surfactant. The activeingredient typically comprises from 0.1 to 100 w/w of the formulation.

In addition to the ingredients specifically mentioned above, theformulations of the present invention may include other agents known tothose skilled in the art of pharmacy, having regard for the type offormulation in issue. For example, formulations suitable for oraladministration may include flavouring agents and formulations suitablefor intranasal administration may include perfumes.

The following Examples are illustrative and should not be interpreted inany way so as to limit the scope of the invention.

EXAMPLES

In general, the potential of a compound of the invention as a drugcandidate can be tested using various assays known in the art. Forexample, for in vitro validation for FXR: its activity and selectivitycan be evaluated using AlphaScreen (biochemical assay); gene expressioncan be evaluated using RT-PCR (FXR target gene); and cytotoxicity (e.g.,HepG2) can be evaluated using ATP content, LDH release, and Caspase-3activation. For the in vitro validation for TGR5: its activity andselectivity can be evaluated using HTR-FRET (cell-based assay); geneexpression can be evaluated using RT-PCR (TGR5 target gene (i.e.,cFOS)); and cytotoxicity (e.g., HepG2) can be evaluated using ATPcontent, LDH release, and Caspase-3 activation. The ADME (absorption,distribution, metabolism, and excretion)/pharmacokinetic properties andin vivo validation of compounds of the invention can also be studiedusing methods known in the art.

Example 1: Synthesis of Compounds 100 and 101

Compounds 100 and 101 were synthesized according to the scheme below.

Reagents and conditions: a) 1) MeOH, p-TSA, ultrasound, 3 h,quantitative; 2) Ac₂O, NaHCO₃, THF, reflux 12 h, 85%; b) PCC, CH₂Cl₂, 6h, 62%; c) Ac₂O, Bi(OTf₃, CH₂Cl₂, 1 h, 91%; d) Br₂, Benzene, 30° C.overnight, 74%; e) NaBH₄, NaOAc, Pyr, r.t. 2 days, 80%; f) HI 57%, AcOH,r.t. 30 min; g) CrO₃, AcOH, r.t. 45 min; h) Zn dust, NaOAc, reflux 20min; i) NaOH 2M, MeOH, r.t. overnight, 65% from compound 5; l) NaBH₄,THF/H₂O 4:1, 70%; m) Na(s), sec-BuOH, 50° C., 70%.

The synthesis is based on the use of 6α-ethyl-cholic acid (6-ECA, 1) asstarting material which was prepared using methods known in the art.6-ECA (1) was treated with p-TSA in MeOH under ultrasound irradiation togive the corresponding methyl ester, which was selectively protected atthe C3 position by refluxing with Ac₂O in the present of NaHCO₃ in THFto afford compound 2. Treating compound 2 with PCC in CH₂Cl₂ at roomtemperature followed by treatment with Ac₂O, Bi(OTf)₃ in CH₂Cl₂ at roomtemperature afforded the intermediate methyl3α,7α-diacetoxy-12-oxo-5β-cholan-24-oate (compound 3; about 48% fromcompound 2).

Treatment of compound 3 with Br₂ in benzene for e.g., 12 h yieldedcompound 4. Reaction of compound 4 with NaBH₄ and NaOAc in freshlydistilled pyridine gave the corresponding 11β-12β epoxide (compound 5),in about 59% yield after silica gel purification. The reaction ofcompound 5 with HI in AcOH at room temperature afforded the halohydrineintermediate which were then oxidized at C11 position with CrO₃ in AcOHto generate compound 6. Reaction of compound 6 with Zn dust in boilingAcOH and alkali hydrolysis (NaOH/MeOH) afforded3α,7α-hydroxy-12-keto-5b-cholan-24-oic acid (compound 7; about 65% yieldfrom compound 5).

Compound 7 was stereoselectively reduced at the C11-carbonyl using NaBH₄in a mixture of THF/H₂O═ (4:1, v/v) to give3α,7α,11β-trihydroxy-6α-ethyl-5β-cholan-24-oic acid (Compound 100; about27% from compound 3), after chromatographic purification to affordCompound 100. Alternatively, compound 7 was reduced with sodium insec-BuOH at 50° C. to give Compound 101 (about 70% yield), afterpurification.

Example 2: Compound 100 is a Potent, Specific FXR Agonist

In the nucleus, ligand-bound nuclear receptors (NRs) modulate initiationof transcription by directly interacting with the basal transcriptionalmachinery or by contacting bridging factors called coactivators (Onate SA, et al., Science 1995; 270:1354-1357; Wang J C, et al., J Biol Chem1998; 273:30847-30850; Zhu Y, et al., Gene Expr 1996; 6:185-195). Theligand-dependent interaction of NRs with their coactivators occursbetween activation function 2 (AF-2), located in the receptorligand-binding domain (LBD) and the nuclear receptor boxes (NR box)located on the coactivators (Nolte R T, et al., Nature 1998;395:137-143). Several lines of evidence have demonstrated that the LXXLLpeptide sequence present in the NR box represents a signature motif thatfacilitates the interaction of different proteins with the AF-2 region(Heery D M, et al., Nature 1997; 387:733-736; Torchia J, et al., Nature1997; 387:677-684).

AlphaScreen was used with the aim of identify novel modulators by takingadvantage of the bimolecular interaction prevailing between FXR and theLXXLL motif present in the NR box of the steroid receptor coactivator 1(SRC-1).

Human FXR-LBD-GST was incubated with increasing concentrations of theindicated ligands in the presence of biotinylated LXXLL SRC-1 peptide.The AlphaScreen signal increases when the complex receptor-coactivatoris formed. EC₅₀ values were 8.9 μM for chenodeoxycholic acid (CDCA;which is a positive control), 0.16 μM for Compound A, and 0.16 μM forCompound 100. These results are the mean±S.D. of triplicate samples froma representative experiment of three performed. The AlphaScreen assay isa very robust and reproducible assay, as shown by the Z′ factor of 0.84(Zhang J H, et al., J Biomol Screen 1999; 4:67-73). Thus, Compound 100is a highly potent FXR agonist.

Further, the data in the table below show that Compound 100 is selectivefor human FXR and is not active for human TGR5.

TABLE 1 HTR-FRET HTR-FRET AlphaScreen (cAMP) (cAMP) Assay Human TGR5Human TGR5 Human FXR (NCI-H716 cells) overexpression Ref. CDCA = Ref.LCA = Ref. LCA = Compound 15 ± 3 μM 7 ± 3 μM 0.9 ± 0.1 μM Compound 1000.180 ± 0.02  No activity No activity Compound 101 3 ± 2 41.5  CompoundA  0.2 ± 0.018 15 ± 5 Compound B 0.03 0.63 Compound C 175 0.9 

Additionally, using the AlphaScreen assay, it was demonstrated thatCompound 100 specifically activates FXR and does not activate 13 othernuclear receptors involved in the metabolic pathways.

TABLE 2 FXR LXRβ PXR CAR PPARα PPARδ PPARγ Activation ActivationActivation Activation Activation Activation Activation Compound (CDCA =(T0901317 = (SR-12183 = (CITCO = (GW7647 = (GW0742 = (GW1929 =(Reference 10-20 μM) 0.08 μM) 0.062 μM) 0.005 μM) 0.003 μM) 0.004 μM)0.012 μM) standard) EC₅₀ (μM) EC₅₀ (μM) EC₅₀ (μM) EC₅₀ (μM) EC₅₀ (μM)EC₅₀ (μM) EC₅₀ (μM) Compound 0.16 No activity No activity No activity Noactivity No activity No activity A Compound 0.03 No activity No activity44* No activity No activity No activity B Compound 0.16 No activity Noactivity No activity No activity No activity No activity 100 RARα VDR TRPR RXR GR ER Activation Activation Activation Activation ActivationActivation Activation Compound (ATRA = (DiHydroxyVitD3 = (T3 =(Corticosterone = (9cisRA = (Budenoside = (Estradiol = (Reference 0.001μM) 0.005 μM) 0.0001 μM) 0.050 μM) 0.004 μM) 0.0002 μM) 0.001 μM)standard) EC₅₀ (μM) EC₅₀(μM) EC₅₀ (μM) EC₅₀ (μM) EC₅₀ (μM) EC₅₀ (μM)EC₅₀ (μM) Compound No activity No activity No activity No activity Noactivity No activity No activity A Compound No activity No activity Noactivity No activity No activity No activity No activity B Compound Noactivity No activity No activity No activity No activity No activity Noactivity 100 *inverse agonist. Values for compound B taken from RizzoG., et al., Mol Pharm., 2010; 78: 617-630.

FXR activation by Compound 100 was also tested in cell-basedtransactivation assays with the use of HEK293T cell line transientlytransfected with Gal4-FXR-LBD chimera and the (UAS)5-Luc system (FIG. 1). FXR activation by Compound 100 was comparable to that induced bycompound A indicating that these compounds are potent FXR agonists incell-based assays. FIG. 1 is a graph showing the activity of Compound100 in comparison to compound A in a transactivation assay in HEK293Tcells. NT is FXR vector-transfected cells without exposure to compound Aor Compound 100. Values are represented in M.

Bile acids (BAs) modulate not only several nuclear hormone receptors,but are also agonists for the G protein-coupled receptor (GPCR) TGR5(Makishima M, et al., Science 1999; 284:1362-1365; Parks D J, et al.,Science 1999; 284:1365-1368; Maruyama T, et al., Biochem Biophys ResCommun 2002; 298:714-719; Klawamata Y, et al., J Biol Chem 2003;278:9435-9440). Signalling via FXR and TGR5 modulates several metabolicpathways, regulating not only BA synthesis and enterohepaticrecirculation, but also triglyceride, cholesterol, glucose, and energyhomeostasis. To evaluate the capacity of a compound of the invention toactivate TGR5, Compound 100 and other comparison compounds were screenedfor an increase of intracellular cAMP as a read-out for TGR5 activation.Human enteroendocrine NCI-H716 cells constitutively expressing TGR5 wereexposed to increasing concentrations of Compound 100, and intracellularcAMP levels were measured by TR-FRET. Lithocholic acid (LCA) was used aspositive control.

As shown in FIG. 2A, Compound 100 does not induce TGR5 activity in cellsexpressing the receptor physiologically as no change in the level ofintracellular cAMP was observed. To further assess if Compound 100 couldbind TGR5, a clonal cell line over-expressing TGR5 was exposed todifferent concentrations of Compound 100. The results illustrated inFIG. 2B show that even with the over-expression of the TGR5 receptor,Compound 100 had no relevant effect. FIG. 2A is a graph showing the TGR5activity of Compound 100 (no activity) and LCA in human enteroendocrinecells expressing TGR5 at physiological level. Results are shown as themean±S. D. of triplicate samples from a representative experiment ofthree performed. FIG. 2B is a graph showing the TGR5 activity ofCompound 100 (no activity) and LCA in human Chinese hamster ovary (CHO)cells over-expressing TGR5.

Example 3: FXR Target Genes Modulated by Compound 100

To evaluate the capacity of Compound 100 to modulate FXR target genes,quantitative RT-PCR assays were performed. HepG2 cells were selected asa relevant cell line to determine whether a compound of the inventioncan regulate the endogenous FXR genetic network. The ability of acompound of the invention to induce FXR target genes was assessed byisolating total RNA from cells treated overnight with 1 μM of compoundsA, B, and 100. Compound A is established as a potent FXR selectiveagonist and compound B is established as a dual potent FXR/TGR5 agonist.Compound 100's gene activation profile in HepG2 cells was compared tothe profiles of compounds A and B. (Pellicciari, R, et al., J Med Chem.2002; August 15; 45: 3569-72; Rizzo, G, et al., Mol. Pharm., 2010; 78:617-630).

FXR regulates the expression of several target genes involved in BAhomeostasis. Briefly, FXR plays a central role in several metabolicpathways, including i.e., lipid metabolism, bile-acids metabolism, andcarbohydrate metabolism. Regarding gene expression profiling, the genesencoding proteins involved in lipid metabolism include, e.g., APOCII,APOE, APOAI, SREBP-1C, VLDL-R, PLTP, and LPL; the genes encodingproteins involved in bile-acids metabolism include, e.g., OSTα/β, BSEP,MRP2, SHP, CYP7A1, FGF19, SULT2A1, and UGT2B4; and the genes encodingproteins involved in carbohydrate metabolism include, e.g., PGC1a,PEPCK, and GLUT2.

As shown in FIGS. 3A-3H, Compound 100 activation of FXR indirectlyrepresses the expression of the BA biosynthetic enzymes CYP7A1 byincreasing the levels of the nuclear receptor SHP in the liver andintestine and increasing the level of FGF19 (Goodwin, B, et al., Mol.Cell 2000; 6: 517-526). Compound 100 activated FXR also positivelyregulates the expression of genes encoding proteins involved in thetransport of BA, including, BSEP, and OSTα and OSTβ. Newly synthesizedBAs are conjugated with taurine or glycine and then actively secreted inthe gall bladder, FXR regulates both of these critical processes.Monoanionic- and dianionic-conjugated BAs are then actively secreted inthe gall bladder by BSEP and the multidrug related protein 2 (MRP2),respectively. These transporters belonging to the ABC transporter familyand are both induced by FXR at the transcriptional level. The regulationof these ABC transporters is of critical importance in order to avoid BAaccumulation in the liver and consequent hepatic injury (Schinkel A H,et al., Mammalian drug efflux transporters of the ATP binding cassette(ABC) family: an overview. Adv Drug Deliv Rev. 2012; September 13).

FIG. 3 are a series of graphs showing the activity of Compound 100 andother comparison compounds in regulating expression of OSTα (A), OSTβ(B), BSEP (C), MRP2 (D), CYP7A1 (E), SHP (F), FGF-19 (G), and UGT2B4(H). Note in the FIGS. 3A-3H, the y-axis displays folds change inexpression relative to untreated cells. The data were normalizedrelative to B2M. The error bars display the standard error of the threereplicates.

FXR activation contributes to reverse cholesterol transport, a processthat results in the delivery of cholesterol from peripheral tissues tothe liver for biliary disposal and consequent fecal elimination(Lambert, G, et al., J Biol Chem 2003; 278, 2563-70). In this metabolicscenario, FXR regulates the expression of phospholipids transfer protein(PLTP), responsible for the transfer of phospholipids and cholesterolfrom LDL to HDL, hepatic lipoproteins, such as ApoE, ApoC-I, ApoC-IV,and scavenger receptor B1(SRB1), which is involved in the hepatic uptakeof HDL.

FXR controls triglyceride (TG) metabolism by regulating hepatic de novolipogenesis and triglyceride clearance. Upon activation by Compound 100,FXR down regulates the expression of SREBP-1c, a transcription factorthat plays a critical role in stimulating fatty acid synthesis andlipogenesis (FIGS. 4A-4D) (Landrier, J F, et al., J Clin Invest 2004;113, 1408-18). In addition to the reduction of de novo lipogenesis, FXRactivation also modulates TG clearance. This additional TG-loweringeffect of FXR is explained at the molecular level by the induction ofkey genes, such as Apo-C-I1 LPL and VDL receptor (Kast, H R, et al., MolEndocrinol 2001; 15, 1720-8).

FIG. 4 are a series of graphs showing the activity of Compound 100 andother comparison compounds in regulating PLTP involved in lipidmetabolism (A), SREBP-1C (B), APOCII (C), and PPARγ (D). Note in theFIGS. 4A-4D, the y-axis displays folds change in expression relative tonot treated cells. The data were normalized relative to B2M. The errorbars display the standard error of the three replicates.

FXR may also have a role in carbohydrate metabolism. (Ma K, et al., JClin Invest. 2006; 116:1102-9). PEPCK gene regulation was studied (FIG.5 ) using Compound 100. FIG. 5 is a graph showing the regulation ofCompound 100 and other comparison compounds on PEPCK gene. The y-axisdisplays folds change in expression relative to not treated cells. Thedata were normalized relative to B2M. The error bars display thestandard error of the three replicates.

Collectively the gene expression studies showed that Compound 100modulates the same FXR target genes as compound A or B (also see Table3).

TABLE 3 Compound A Compound B Compound 100 gene (1 μM) (1 μM) (1 μM)OSTα up up up OSTβ up up up BSEP up up up SHP up up up CYP7α1 down downdown UGT2B4 up up up MRP2 up up up FGF-19 up up up PPARγ up up up PLTPup up up APOCII up up up PEPCK up up up SREBP-1C down down down

Example 4: Compound 100 does not Exert Cytotoxic Effects in HepG2 Cells

To evaluate in vitro cytotoxicity of Compound 100, two different assaymethods were employed. The assays evaluated cell viability by measuringATP levels and cytotoxicity by measuring LDH release. AdenosineTriphosphate (ATP) nucleotide represents the source of energy at thebasic molecular level, as it is a multifunctional molecule that is usedin every cell as a coenzyme and is an integral part of the mitochondrialDNA (Kangas L, et al., Medical Biology, 1984; 62, 338-343; Crouch S P M,et al., J. Immunol. Methods, 1993; 160, 81-88; Petty R D, et al., J.Biolumin. Chemilumin. 1995; 10, 29-34). It has been called the“molecular unit of currency” when it comes to intracellular energytransfer. This is to ensure the important role of ATP in metabolism anda drop in ATP content is the first step in revealing cellular damage(Storer R D, et al., Mutation Research, 1996; 368, 59-101; Cree I A,Andreotti P E., Toxicology in Vitro, 1997; 11, 553-556).

Cell viability was determined as measure of intracellular ATP related tothe time of exposure and concentration of the test compounds (Sussman, NL.; Promega Cell Notes, Issue 3. 2002).

FIG. 6 is a graph showing the measurement of ATP in HepG2 cells, treatedwith the indicated concentrations of compounds for 4 h. It demonstratedthat all cells in presence of different concentrations of Compound 100were viable as cells treated with the vehicle alone, i.e., all cellstreated with Compound 100 remain viable (100%). LCA, a well-knowncytotoxic bile acid, was used as comparator and Tamoxifen was used aspositive controls for the assays.

An additional method to determine the viability of cells is to detectthe integrity of the membrane that defines the cellularcompartmentalization. Measuring the leakage of components out of thecytoplasm, in damaged cell membranes, indicates loss of membraneintegrity, and LDH release is the method used to determine commontoxicity in cells. HepG2 cells were treated with Compound 100, serialdilutions were performed, LCA dilutions were added to the plated cellsas assay control together with no-cell and untreated cells. The assaywas performed in triplicate for each test compound concentration.

The results show that Compound 100 does not induce any cytotoxic effecton HepG2 cells. Lithocolic Acid increased LDH release at 70 μM whilstthe control Tamoxifen exerted the cytotoxic effects at approximately 25μM (see Table 4).

TABLE 4 Membrane integrity EC₅₀ (μM) Compound (LDH measure) Tamoxifen 35 ± 10 LCA 75 ± 5 Compound A 190 ± 30 Compound B* 670 Compound 100 Notoxicity (100% living cells) Compound 101 No toxicity (100% livingcells) *Rizzo et al., Mol. Pharm. 2010

Example 5: Compound 100 does not Inhibit Cytochrome P450 Enzymes

To evaluate the potential of Compound 100 for drug-drug interactions,the six main CYP450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1,CYP3A4) were investigated. (Obach, R S, et al., J Pharmcol Exp Ther,2006; 316(1): p. 336-48).

To determine interaction between Compound 100 and cytochrome P450enzymes, Compound 100 was analyzed by its capacity to inhibit (or not)the production of a fluorescent signal, using recombinant CYP450proteins (baculosomes; Invitrogen), substrates and inhibitors (Bidstrup,T B, et al., Br J Clin Pharmacol, 2003; 56(3): p. 305-14). As a positivecontrol, a selective inhibitor for each CYP450 isoform was tested in thesame plate (Table 5).

TABLE 5 Compound A Compound B Compound 100 CPY450 IC₅₀ (μM) IC₅₀ (μM)IC₅₀ (μM) CYP1A2 >10 >10 >10 Reference: Furafylline = 0.5 μM CYP3A4(Green >10 >10 >10 Substrate) Reference: Ketoconazole = 0.044 μM CYP3A4(Blue >10 >10 >10 Substrate) Reference: Ketoconazole = 0.04 μMCYP2C9 >10 >10 >10 Reference: Sulfaphenazole = 0.4 μMCYP2C19 >10 >10 >10 Reference: Miconazole = 0.06 μM CYP2D6 >10 >10 >10Reference: Quinidine = 0.01 μM CYP2E1 >10 >10 >10 Reference: DCC = 0.4μM IC₅₀ > 10 μM means that the compound does not inhibit the CYP450. Theresults obtained demonstrated that Compound 100, like compounds A and B,does not inhibit the Cytochrome P450 enzymes tested, showing thatCompound 100 is not likely to be influenced by drug-drug interactioneffects. (Rizzo, G, et al., Mol Pharm, 2010; 78: 617-630).

Example 6: Compound 100 does not Inhibit Human ERG Potassium Channel

To determine ion channel function, Predictor™ hERG FluorescencePolarization assay was employed as it provides an efficient method foran initial determination of the propensity of test compounds to blockthe hERG channel (Dom, A, et al. J Biomol Screen, 2005; 10(4): 339-47).The assay is based on the assumption that the hERG potassium channelactivity contributes to the resting membrane potential in permanentlytransfected cells, and thus a block of hERG channels should result in adepolarization of the cell membrane. The assay was designed to identifypotential hERG channel blockers by producing data that accuratelycorrelates with patch-clamp electrophysiology studies. Results from thePredictor assay demonstrate a high correlation with those obtained frompatch clamp techniques (Table 6) (Dom, A, et al. J Biomol Screen, 2005;10(4): 339-47).

TABLE 6 Patch-Clamp* Radioligand* FP Compound IC₅₀ (nM) Astemizole 1.2 11.3 Dofetilide 12 40 6.9 Terfenadine 16 30 23 E-4031 48 20 34 Bepridil550 170 210 Thioridazine 1250 510 708 Fluoxetine 990 2230 4310Amitripyline 10000 f2440 11200

Table 6 show the comparison of IC₅₀ values generated with the Predictor™hERG Fluorescence Polarization assay with reported IC₅₀ values frompatch-clamp and radioligand displacement assays.

Membrane preparations from Chinese hamster ovary cells stablytransfected with hERG potassium channel were used to evaluate thepotential inhibitory effect of Compound 100 on this channel using thePredictor fluorescence polarization assay. Reduction of membranepolarization as a result of inhibition of the hERG potassium channel isdirectly correlated with a reduction of the fluorescence polarization(FP). The results show that like compounds A and B, Compound 100 doesnot block or inhibit the hERG potassium channel.

The assay was performed in triplicate by using a 16-point dose-responseof test compound and the positive controls E-4031 and Tamoxifen. An IC₅₀of 15 nM (ΔmP=163) for E-4031 and 1.4 μM (ΔmP=183) for Tamoxifen wereobtained. The assay window more than 100 mP (millipolarization) isconsidered good. Z′ value was 0.78 indicates an excellent assay. Thenon-linear regression curves were obtained by GraphPad Prism (GraphPadSoftware Inc.) analysis, to calculate the IC₅₀ values.

Briefly, signalling through FXR modulates a variety of metabolicpathways, so selective FXR modulators are attractive candidates for thetreatment of a range of chronic diseases affecting liver, kidney, aswell as metabolic diseases. Results in the examples described hereincharacterize Compound 100, as a potent and specific FXR agonist.

Remarkably, although it potently activated FXR, Compound 100 showed noactivity against other nuclear receptors and did not active the bileacid GPCR TGR5. In addition to high nuclear receptor selectivity,Compound 100 possesses a pharmacological profile suitable for a drugcandidate. Compound 100 shows no cytotoxic effect on human HepG2 livercells, indicating a lack of liver toxicity, and does not inhibit any ofthe CYP450 enzymes tested, indicating that Compound 100 is devoid ofsignificant drug-drug interaction risk. Further, Compound 100 does notinhibit the human ERG potassium channel.

The combined selectivity and potency of Compound 100 together with itsfavorable drug-like properties, in particular an excellent safetyprofile, make Compound 100 an attractive candidate for treating andpreventing disease.

Example 7: Physiochemical Properties of Compound 100

Physiochemical properties of Compound 100 such as water solubility,critical micellular concentration, surface tension and Log P_(A) weredetermined using methods known in the art. These properties of Compound100 were compared with natural and synthetic analogues (Table 7).

TABLE 7 Ws^((a)) CMC^((b)) ST_(CMC) ^((c)) Bile Acid (μM) (mM) (Dyne/cm)LogP_(A−) ^((d)) Compound 100 143-150 15.8  47.8 0.8 CA 273 9-11 49.01.1 CDCA 32 3.2 45.5 2.2 UDCA   7-7.5 6-10 50.5 2.2 TCDCA hs 3.0 — 0.9TUDCA hs 2.2 — 1.1 Compound A 9 2.9 43.2-48.8 2.5 Compound B hs 1.343.3-47.9 2.0 Compound C 99 2   50.1 1.4 Compound D 15 — — 2.9 CompoundE 120 5.9 52.4 1.6 ^((a))Ws: water solubility refers to BA as protonatedspecies and therefore not evaluated for Compound B, TCDCA and TUDCAwhich are highly soluble (hs); ^((b))CMC: Critical MicellarConcentration determined in 0.15M NaCl water solution; ^((c))ST_(cmc):Surface Tension at CMC in 0.15M NaCl water solution; ^((d))LogP_(A) ⁻ :1-octanol-water partition coefficient of the studied bile acids asionized species;

Example 8: Pharmacokinetics and Metabolism in Bile Fistula Rat after idand iv Administration: In-Vivo

The in-vivo models, rats, were administered single dose of Compound 100at 1 μmol/min/Kg·1 hour (see FIGS. 7A, 7B, and 7C). FIG. 7A is a graphshowing the choleretic effect of Compound 100 for id and ivadministration. FIG. 7B is a graph showing the secretion of Compound 100over time for id and iv administration. FIG. 7C is a graph showing theplasma concentration of Compound 100 over time.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the present invention.

All patents, patent applications, and literature references cited hereinare hereby expressly incorporated by reference.

1. A method of treating fibrosis or one or more symptoms of intrahepaticcholestasis or extrahepatic cholestasis in a subject in need thereof,comprising administering to the subject an effective amount of acompound of formula I:

or a pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof, wherein: R¹ is hydroxyl; R² is hydrogen, hydroxyl, alkyl, orhalogen, wherein said alkyl is unsubstituted or substituted with one ormore R^(a); R³ is hydrogen, hydroxyl, alkyl, or halogen, wherein saidalkyl is unsubstituted or substituted with one or more R^(b); R⁴ isalkyl, alkenyl, alkynyl, or halogen, wherein said alkyl is unsubstitutedor substituted with one or more R^(c); R^(a), R^(b), and R^(c) are eachindependently halogen or hydroxyl; R⁵ is hydroxyl, OSO₃H, OSO₃ ⁻,OCOCH₃, OPO₃H₂, OPO₃ ²⁻, or hydrogen; and R⁶ is hydroxyl, OSO₃H, OSO₃ ⁻,OCOCH₃, OPO₃H₂, OPO₃ ²⁻, or hydrogen; or taken together R⁵ and R⁶ withthe carbon atom to which they are attached form a carbonyl.
 2. Themethod of claim 1, having the formula:

or a pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof.
 3. The method of claim 1, wherein one of R² or R³ is hydroxyland the remaining R² or R³ is hydrogen.
 4. The method of claim 1,wherein one of R⁵ or R⁶ is hydroxyl and the remaining R⁵ or R⁶ ishydrogen.
 5. The method of claim 1, wherein R² is hydroxyl and R³ ishydrogen.
 6. The method of claim 1, wherein R⁵ is hydroxyl and R⁶ ishydrogen.
 7. The method of claim 1, wherein R² and R⁵ are each hydroxyland R³ and R⁶ are each hydrogen.
 8. The method of claim 1, wherein R⁴ isalkyl.
 9. The method of claim 1, wherein R⁴ is unsubstituted alkyl. 10.The method of claim 1, wherein R⁴ is ethyl.
 11. The method of claim 1,having the following formula:

or a pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof.
 12. The method of claim 1, for treating fibrosis.
 13. Themethod of claim 1, for treating one or more symptoms of intrahepaticcholestasis or extrahepatic cholestasis.
 14. The method of claim 12,wherein the fibrosis is fibrosis due to pathological conditions ordiseases, fibrosis due to physical trauma, fibrosis due to radiationdamage, fibrosis due to exposure to chemotherapeutics, liver fibrosis,fibrosis of the kidneys, fibrosis of the lung, or fibrosis of theintestine.
 15. The method of claim 12, wherein the fibrosis is due to adisease selected from primary biliary cirrhosis, primary sclerosingcholangitis, fatty liver, obesity, non-alcoholic steatohepatitis, cysticfibrosis, hemochromatosis, auto-immune hepatitis, and steatohepatitis.16. The method of claim 12, wherein the fibrosis is fibrosis secondaryto surgical scarring, accidental physical trauma, burns, or hypertrophicscarring.
 17. The method of claim 13, wherein the one or more symptomsof intrahepatic cholestasis or extrahepatic cholestasis is biliaryatresia, obstetric cholestasis, neonatal cholestasis, drug inducedcholestasis, or cholestasis arising from Hepatitis C infection.